animals
Article
Characterization of Testicular Tumor Lesions in Dogs by
Different Ultrasound Techniques
Riccardo Orlandi 1 , Emanuela Vallesi 1,2 , Cristiano Boiti 3 , Angela Polisca 4, *, Paolo Bargellini 1
and Alessandro Troisi 5






Citation: Orlandi, R.; Vallesi, E.; Boiti,
C.; Polisca, A.; Bargellini, P.; Troisi, A.
Characterization of Testicular Tumor
Lesions in Dogs by Different
Ultrasound Techniques. Animals 2022,
12, 210. https://doi.org/10.3390/
ani12020210
Academic Editors: Marco Quartuccio,
Massimo De Majo and Santo
Cristarella
Received: 15 November 2021
Accepted: 13 January 2022
Published: 17 January 2022
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
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iations.
Copyright: © 2022 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
Animals 2022, 12, 210. https://doi.org/10.3390/ani12020210
1 Anicura Tyrus Clinica Veterinaria, Via Bartocci 1G, 05100 Terni, Italy; riccardo.orlandi@anicura.it (R.O.);
emanuela.vallesi@anicura.it (E.V.); paolo.bargellini@anicura.it (P.B.)
2 Anicura CMV Clinica Veterinaria, Via G.B. Aguggiari 162, 21100 Varese, Italy
3 Tyrus Science Foundation, Via Bartocci 1G, 05100 Terni, Italy; boiti.cristiano@gmail.com
4 Department of Veterinary Medicine, University of Perugia, Via San Costanzo 4, 06126 Perugia, Italy
5 School of Biosciences and Veterinary Medicine, University of Camerino, Via Circonvallazione 93/95,
62024 Macerata, Italy; alessandro.troisi@unicam.it
* Correspondence: angela.polisca@unipg.it; Tel.: +39-07-5585-7623
Simple Summary: In the present work, we investigated the hypothesis that testicular tumor lesions
of dogs could present a specific vascular pattern, which could be detected and differentiated by
distinctive ultrasonography techniques. This is a relevant issue since it could contribute to determine
their etiology based on the different vascular patterns before dogs undergo surgery. To this end, we
implemented a multiple ultrasonographic approach consisting of B-Mode ultrasonography, color
Doppler ultrasound, B-flow, and contrast enhanced ultrasound in 27 dogs with different testicular
tumors, including leydigomas (n = 14), seminomas (n = 8), sertoliomas (n = 6), and mixed cells (n = 5).
B-Mode ultrasonography did not differentiate tumor types. Pulsatility and resistive indexes of
pampiniform and testicular arteries as assessed by pulse Doppler as well as the presence perilesional
and/or perilesional/intralesional blood flow patterns as assessed by color and pulsed Doppler and
B-flow differed between tumor types. In conclusion, despite the limited number of cases, we found
that testicular tumors of dogs have subtly different vascular patterns. These architectural details are
enhanced by multiparametric ultrasonography, which is highly recommended for the identification
of their etiology.
Abstract: In this retrospective study, we assessed the accuracy of different blood flow imaging in
diagnosing testicular tumor types in dogs. We recruited 27 dogs with leydigomas (14), semino-
mas (eight), sertoliomas (six), and mixed cells (five) confirmed histopathologically. In intact dogs,
Pampiniform plexus and marginal arteries were scanned through pulsed Doppler. Blood flow and
presence of intralesional/perilesional arteries were assessed by color and power Doppler, B-flow, and
contrast-enhanced ultrasound. Tumor types did not differ by B-Mode ultrasonography characters.
Pampiniform and testicular arteries of sertoliomas had higher (p < 0.05) pulsatility and resistive
indexes. The proportion of leydigomas with a perilesional and/or perilesional/intralesional blood
flow pattern detected by color and pulsed Doppler and B-flow was higher (p < 0.05) than that of the
other tumors counted together. This resulted in a sensitivity of 81.8%, 83.3%, and 85.7%, a specificity
of 76.5%, 56.3%, and 73.7%, and a correct classification rate of 78.6%, 67.9%, and 78.8%, respectively.
While contrast enhanced ultrasound was highly effective in detecting all tumors, qualitative and
quantitative parameters did not contribute to their differential diagnosis. In conclusion, results indi-
cate that different testicular tumor types of dogs have subtly different vascular patterns, a condition
that could help in identifying leydigomas.
Keywords: testes; ultrasonography; CEUS; color Doppler; B-flow; canine tumors
https://www.mdpi.com/journal/animals
Animals 2022, 12, 210 2 of 17

1. Introduction
Testicular tumors represent the most common tumors of the canine male genitalia with
a prevalence that ranges from 2% to 60% and an incidence that increases with age [1–3].
Among the testicular tumors, seminomas, Sertoli cell tumors, and Leydig cell tumors are
the most common [4].
For many years, two-dimensional ultrasonography has been part of routine inves-
tigation of the male reproductive tract in both human and veterinary medicine [5,6]. It
represents the imaging technique of choice for the detection and characterization of testic-
ular lesions. Even though this diagnostic approach can help in differentiating neoplastic
processes from other pathologies, such as orchitis, testicular torsion, and epididymitis,
the ultrasonographic changes are not specific enough to identify the different types of
testicular tumors [7,8]. In fact, the ultrasonographic features of testicular tumors are ex-
tremely variable [9,10]. Therefore, once detected, testicular tumor-like lesions require
further characterization.
Recently, color Doppler ultrasonography and contrast enhanced ultrasound (CEUS)
have been applied in in human males and dogs to improve the diagnostic accuracy of
testicular tumors and help to decide whether sparing surgery or total orchiectomy was
indicated [7,11–14]. In fact, two-dimensional ultrasonography offers valuable, but only
structural information about testicular tumors, while color Doppler and CEUS provide
information about their real time blood flow [15]. This information is clinically relevant be-
cause vascularization and micro-vessel density, i.e., the number of vessels per mm2 , reflect
tumor angiogenesis and are related to its malignancy [16,17]. Many studies have shown
that angiogenesis promotes tumor growth by supplying essential oxygen and nutrients
to neoplastic cells [17–20]. At the end of the last decade, the development of the B-flow
technique with high-frequency transducers allowed a non-Doppler visualization of blood
flow imaging [21] that was considered superior for evaluating complex hemodynamics of
peripheral vascular pathologies in human males [22].
It is therefore not surprising that a multiparametric approach consisting of conven-
tional ultrasonography, color Doppler ultrasound, and CEUS is increasingly being used
in humans for differentiating benign from malignant intra-testicular lesions [15]. On the
contrary, as per our knowledge, such a multiparametric approach has never been tested in
the small animal practice. Indeed, the use of reliable diagnostic tools would be relevant for
the detection and characterization of testicular tumors that can compromise fertility, espe-
cially in stud dogs. We focused our attention on breeding dogs to evaluate the possibility
of preserving their fertility. In addition, an early diagnosis of a testicular tumor can also
reduce the risk of metastasis, even if that occurs less frequently in dogs than humans [23].
Therefore, in the present study, we evaluated the diagnostic accuracy of two-dimensional
ultrasonography combined with Doppler ultrasound, B-flow imaging, and CEUS in the
assessment of intra-testicular tumor lesions in dogs. We assumed that such a multiparamet-
ric ultrasound approach would further improve the diagnostic and descriptive potential of
testicular lesions, while also trying to evaluate malignancy as an increase in the vascularity
of testicular lesions.

2. Materials and Methods

2.1. Case Selection
All dogs included in this prospective study were presented to the Tyrus Veterinary
Clinic from January 2016 to May 2018. In all cases, we obtained informed owner consent
prior to enrolling the dogs in the study. We reviewed results for 27 consecutive dogs
with complete medical records and ultrasonographic evidence of testicular tumor lesions
supported by histopathological diagnoses following orchiectomy. Clinical data included
signaling, medical history, physical findings, as well as routine hematological and biochem-
ical analyses. The diagnosis of a suspected testicular tumor-like lesion was based on initial
clinical findings and/or ultrasonographic images. Once suspected, abdominal ultrasound
examination and chest X-ray were also performed. After conventional B-mode ultrasound
 on initial clinical findings and/or ultrasonographic images. Once suspected, abdominal
ultrasound examination and chest X-ray were also performed. After conventional
Animals 2022, 12, 210 B-mode ultrasound examination, all testicles were evaluated by color and spectral 3 of 17
Doppler vascular ultrasonography, power Doppler, B-flow, and CEUS. After surgical
intervention, all testicular lesions were examined histopathologically for a definitive
diagnosis. all testicles were evaluated by color and spectral Doppler vascular ultrasonog-
examination,
raphy,Exclusion criteriaB-flow,
power Doppler, were and
the CEUS.
presence of surgical
After concomitant pathologies,
intervention, anamnesis
all testicular lesionsof
chronic diseases and/or acute illnesses in the last year,
were examined histopathologically for a definitive diagnosis.uncompleted diagnostic panel, or
histopathological diagnosis
Exclusion criteria notpresence
were the confirming the presencepathologies,
of concomitant of a testicular tumor lesion.
anamnesis of chronic
diseases and/or acute illnesses in the last year, uncompleted diagnostic panel, or histopatho-
2.2. Ultrasound
logical diagnosis Procedures and Image
not confirming the Analysis
presence of a testicular tumor lesion.
All sonographic examinations were performed by the same operators using a
2.2.
LOGIQUltrasound Procedures
E9 scanner and Image
(GE Medical Analysis
Systems, Milwaukee, WI, USA) equipped with a (9L GE
All sonographic
Medical examinations
Systems, Milwaukee, WI, were
USA)performed
2.5–8 MHzby the same
linear probeoperators using dogs
on unsedated a LOGIQ
held
E9 scannerrecumbency.
in lateral (GE MedicalInSystems,
dogs with Milwaukee, WI, USA)lesions,
multiple testicular equippedwe with a (9Ldata
recorded GE Medical
for each
Systems,
lesion. ForMilwaukee, WI, USA)
all ultrasound methods2.5–8 MHzthe
used, linear probe on unsedated
characteristics dogswere
of the lesions heldcompared
in lateral
recumbency.
to normal surrounding testicular parenchyma, whilst in the case of lesions involvingFor
In dogs with multiple testicular lesions, we recorded data for each lesion. the
all ultrasound
whole gonad methods
the normal used, the characteristics
parenchyma of the lesionstesticle
of the contralateral were compared to normal
was considered to
surrounding
compare data. testicular parenchyma, whilst in the case of lesions involving the whole gonad
the normal parenchyma
Longitudinal of the contralateral
and transverse views were testicle was considered
obtained from eachtotestis
compare
usingdata.
B-mode.
Longitudinal
B-Mode ultrasound and transverseincluded
parameters views were obtained
anatomical from
site each testis
of affected using(scrotal
testicle B-mode.or
B-Mode ultrasound parameters included anatomical site of affected
abdominal), maximal diameter (cm), location (left, right, or both testicle), margins testicle (scrotal or
abdominal), maximal diameter
(regular, irregular), (cm), location
echogenicity (left, right,
(hyperechoic, or both testicle),
hypoechoic, margins
isoechoic, (regular,
anechoic or
irregular), echogenicity (hyperechoic, hypoechoic, isoechoic, anechoic
mixed), and echotexture (homogeneous or inhomogeneous) of the lesions relative or mixed), and echo-
to
texture
normal(homogeneous
surrounding or(or inhomogeneous)
contralateral, of inthe lesions
the caserelative
of a to normallesion)
diffuse surrounding (or
testicular
contralateral, in the case
parenchyma (Figure 1). of a diffuse lesion) testicular parenchyma (Figure 1).

Figure 1.1.Representative
Figure RepresentativeB-Mode
B-Mode ultrasonography
ultrasonography scans.
scans. (A)(A)
LeftLeft
testistestis of year
of a 11 a 11 old
year old mixed
mixed breed
breed
dog doga with
with focalahyperechoic
focal hyperechoic
nodulenodule (seminoma)
(seminoma) characterized
characterized by homogenous
by homogenous echotexture
echotexture and
and regular margin. (B) Mixed cell tumor (leydig and seminoma) recorded as two
regular margin. (B) Mixed cell tumor (leydig and seminoma) recorded as two nodular lesions in nodular lesions
in the right testis of a 13 year old Epagneul Breton. The lesions had mixed patterns, inhomogeneous
the right testis of a 13 year old Epagneul Breton. The lesions had mixed patterns, inhomogeneous
echotextures and irregular margins.
echotextures and irregular margins.

2.3. Color, Pulsed, and Power Doppler

The testicular artery was first detected in the pampiniform plexus by scanning the
cranial pole of the testis. Then, the linear probe was moved towards the ventral region of
testis to locate the marginal artery. Finally, the ultrasound beam was oriented to optimize
 Animals 2022, 12, x FOR PEER REVIEW 4 of 17

2.3. Color, Pulsed, and Power Doppler

Animals 2022, 12, 210 The testicular artery was first detected in the pampiniform plexus by scanning the 4 of 17
cranial pole of the testis. Then, the linear probe was moved towards the ventral region of
testis to locate the marginal artery. Finally, the ultrasound beam was oriented to optimize
thevisualization
the visualizationof of any
any lesions
lesions present
presentand andcharacterize
characterizetheir
theirblood
bloodflow. Based
flow. on on
Based color
color
Doppler imaging, the blood flow of the lesion was classified as either absent
Doppler imaging, the blood flow of the lesion was classified as either absent or present and, or present
inand,
suchinasuch
case,aascase, as peripheral
peripheral or intralesional
or intralesional (Figure (Figure 2A). Using
2A). Using pulsedpulsed
wavewave
mode, mode, a
a sample
sample volume was successively placed on the pampiniform plexus
volume was successively placed on the pampiniform plexus as well as marginal and lesionas well as marginal
and lesion
arteries arteriesto(if
(if present) present)
record to record the
the waveforms of atwaveforms
least threeof at least three
consecutive consecutive
cardiac cycles. The
cardiac cycles. The PW was set as follows: frequency 3.1 MHz, pulse repetition frequency
PW was set as follows: frequency 3.1 MHz, pulse repetition frequency 3.3 kHz, and Gain
3.3 kHz, and Gain 41 dB. The sample volume, the region where the vessel was studied,
41 dB. The sample volume, the region where the vessel was studied, was positioned in the
was positioned in the artery that allowed the best insonation angle when both peri and
artery that allowed the best insonation angle when both peri and intralesional vessels were
intralesional vessels were detected simultaneously in the same lesion. The insonation
detected simultaneously in the same lesion. The insonation angle between the Doppler
angle between the Doppler stream and the course of the vascular segment was manually
stream and the course of the vascular segment was manually aligned and the measured
aligned and the measured blood flow velocity was automatically corrected. The
blood flow velocity was automatically corrected. The measurements with an angle >20◦
measurements with an angle >20° were disregarded. However, the measurements of
were disregarded. However, the measurements of three different waveforms in each vessel
three different waveforms in each vessel were taken and we made an average to reduce
were
any taken
errors.and
For weeachmade an average
testicular artery, to reduce anyblood
quantitative errors.
flowFor each testicular
analysis included artery,
the
quantitative
evaluation of blood
peakflow analysis
systolic included
velocity the evaluation
(PSV), end of peak
diastolic velocity systolic
(EDV), velocity
resistance (PSV),
index
end diastolic
(RI), velocityindex
and pulsatility (EDV),(PI)resistance
(Figure 2B).index (RI), and pulsatility index (PI) (Figure 2B).

Figure2.2.Representative
Figure Representative Doppler
Doppler characterization
characterization ofofaanodular
nodularlesion
lesion(leydigoma) found
(leydigoma) in the
found leftleft
in the
testicle of a 11 year old Italian Mastiff. In particular, picture 2 (A), recorded by color Doppler,
testicle of a 11 year old Italian Mastiff. In particular, picture 2 (A), recorded by color Doppler, allowed
allowed the visualization of blood flow distributed peripherally to the lesion, while picture 2 (B)
the visualization
showed the sameofblood
bloodflow
flowsampled
distributed peripherally
by pulsed wave modeto theforlesion, while picture
quantitative 2 (B) showed the
analysis.
same blood flow sampled by pulsed wave mode for quantitative analysis.
Using power Doppler, the blood flow of the testicular lesions was color mapped and
Using as
classified power Doppler,
for color Dopplerthewhen
blood flow of
present. the testicular
Doppler lesions
signals were thenwas color mapped
quantified by a
and
computer-assisted image analysis system using open-source software quantified
classified as for color Doppler when present. Doppler signals were then (ImageJ
byhttp://rsbweb.nih.gov/ij/,
a computer-assisted image analysis
accessed on 15system
November using open-source
2021) to measuresoftware
the total (ImageJ
number of http:
//rsbweb.nih.gov/ij/,
color pixels. accessed on 15 November 2021) to measure the total number of
color pixels.
2.4. B-Flow Examination
2.4. B-Flow Examination
B-flow examination was focused of the vascularization on the testicular tumor
B-flow
lesions. Onceexamination was focusedwas
testicular parenchyma of the vascularization
visualized by B-mode,on the
the testicular
system was tumor lesions.
switched
Once
to B-flow. Still images and video clips were saved for each location examined B-flow.
testicular parenchyma was visualized by B-mode, the system was switched to for
Still images and
subsequent video clips
analyses. were saved
The vessels of thefor eachlesions
tumor location examined
were scored for subsequent
as 0, analyses.
1, or 2 if absent,
The vessels
smaller, or of the than
larger tumor lesions
2 mm were scored
in diameter, as 0, 1, or
respectively. 2 if absent,
Moreover, thesmaller, or larger
vascularization wasthan
2 classified
mm in diameter,
as P if therespectively.
vessels wereMoreover,
perilesional the
orvascularization was classified
I if distributed within the tumoraslesion
P if the
vessels
(Figurewere
3). perilesional or I if distributed within the tumor lesion (Figure 3).
Animals 2022,12,
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17

Figure 3.3. Conventional

Figure Conventional ultrasonographic
ultrasonographic scans
scans (left
(left panels)
panels) ofof two
two different
different seminomas
seminomas found
found
respectively in the right testicle of a 10 year old Labrador Retriever in (A) and in the right testicle of of
respectively in the right testicle of a 10 year old Labrador Retriever in (A) and in the right testicle a
a 9.5 year old German shepherd in (B). In the right panel the B-flow images show the I distribution
9.5 year old German shepherd in (B). In the right panel the B-flow images show the I distribution
of the lesional vessels (arrow) scored as 2 in (A), while in (B) it was possible to record the P pattern
of the lesional vessels (arrow) scored as 2 in (A), while in (B) it was possible to record the P pattern
of vessels distribution (arrows) scored in this case as 1. In this B-flow image, it is also possible to
of vessels some
visualize distribution (arrows)
physiological scored in
testicular this case
vessels as 1. head),
(arrows In this represented
B-flow image, by itthe
is also possible
marginal to
region
visualize some physiological
and intra-testicular branchestesticular vessels
of testicular (arrows head), represented by the marginal region and
artery.
intra-testicular branches of testicular artery.
2.5. Contrast-Enhanced Ultrasonography
2.5. Contrast-Enhanced Ultrasonography
We used the same linear transducer 9 L (GE Medical Systems, Milwaukee, WI, USA)
We used the same linear transducer 9 L (GE Medical Systems, Milwaukee, WI, USA)
set at a frequency of 2.5 MHz with a 0.09 mechanical index and a 90 dB dynamic range for
set at a frequency of 2.5 MHz with a 0.09 mechanical index and a 90 dB dynamic range for
CEUSevaluation.
CEUS evaluation. For
For each
eachdog,dog,aabolus
bolusdose
dose(0.03
(0.03mL/kg
mL/kg of of body
body weight)
weight) of of the
the freshly
freshly
prepared contrast agent (SonoVue R, Bracco Imaging, Milan, Italy) was rapidly infused via
R
prepared contrast agent (SonoVue , Bracco Imaging, Milan, Italy) was rapidly infused
a three-way
via valve
a three-way valve connected
connected totoanan18/20
18/20GGcatheter
catheterin in the
the cephalic vein. The
cephalic vein. The contrast
contrast
injection was
injection was immediately
immediately followed
followed by by aa 55 mL
mL saline
saline flush
flush(NaCl
(NaCl0.9%),
0.9%), with
withrepeated
repeated
administrations of the contrast medium being used in cases of multiple
administrations of the contrast medium being used in cases of multiple lesions. All CEUS lesions. All CEUS
examinations were performed by the same two operators. The first
examinations were performed by the same two operators. The first operator injected the operator injected the
contrast medium through the catheterized vein, while the second performed
contrast medium through the catheterized vein, while the second performed the US scans the US scans
byholding
by holdingthe
thetransducer
transduceras asstill
stillasaspossible
possibleat atthe
theselected
selectedposition
positionduring
duringthe thecontrast
contrast
study.This
study. Thisprocedure
procedurewas wasperformed
performedin inboth
bothgonads
gonadswithwithan aninterval
intervalof of55min.
min.During
During
each examination,
each examination, aa 1.5
1.5 min
minvideovideoclipclipwas
wasrecorded
recorded simultaneously
simultaneously with
withinjection
injection of the
of
contrast agent and saved in the hard disk of the ultrasound machine
the contrast agent and saved in the hard disk of the ultrasound machine for subsequent for subsequent
analyses.
analyses.
Contrast enhancement
Contrast enhancement patterns
patterns during
during thethe distribution
distribution phases
phases were
werefirst
firstevaluated
evaluated
qualitatively for
qualitatively for each
each testicle
testicle to to assess
assess the
the following
following parameters
parameters compared
compared to to normal
normal
testicular parenchyma:
testicular parenchyma: (i) (i) arrival
arrival time
time ofof contrast
contrast medium
medium into into the
the lesion
lesion (early
(early oror late);
late);
(ii) wash-in
(ii) wash-in enhancement
enhancement (homogeneous
(homogeneous or or inhomogeneous;
inhomogeneous; (iii) (iii) intensity
intensity of of contrast
contrast
(hyperenhanced,isoenhanced,
(hyperenhanced, isoenhanced,or orhypoenhanced)
hypoenhanced)(Figure (Figure4). 4).
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Figure4.
Figure 4. Left
Left testicle
testicle leydigoma
leydigomaof ofaa 11
11 year
year old
old Italian
Italian Mastiff
Mastiff(the
(thesame
sameofofFigure
Figure2).
2).The
Theleft
leftpanel
panel
shows the sagittal B-mode ultrasound highlighting a focal hyperechoic nodule
shows the sagittal B-mode ultrasound highlighting a focal hyperechoic nodule with homogeneouswith homogeneous
echotexture and
echotexture andregular
regularmargin.
margin.Representative
Representative contrast-enhanced
contrast-enhanced ultrasound
ultrasound images
images of
of different
different
contrast distribution phases are represented in the right panels. After 16 s from contrast injection it
contrast distribution phases are represented in the right panels. After 16 s from contrast injection it
is possible to appreciate an early distribution of the contrast within the lesions (Arrows) compared
is possible to appreciate an early distribution of the contrast within the lesions (Arrows) compared
to the surrounding parenchyma (A). Later (25 s) the contrast medium distributes homogeneously
to the surrounding
within the lesion thatparenchyma (A). Later (25compared
remain hyperenhanced s) the contrast
to themedium distributes
normal testicle (B). homogeneously
within the lesion that remain hyperenhanced compared to the normal testicle (B).
Using the software integrated with the ultrasound machine, quantitative assessment
Using the software integrated with the ultrasound machine, quantitative assessment
of contrast enhancement was performed on specific regions of interest (ROIs). In
of contrast enhancement was performed on specific regions of interest (ROIs). In particular,
particular, two ROIs of the same size (or more in case of multiple lesions) were drawn for
two ROIs of the same size (or more in case of multiple lesions) were drawn for each diseased
each diseased testicle: one included the lesion (or the lesions) and the other the normal
testicle: one included the lesion (or the lesions) and the other the normal parenchyma.
parenchyma. In the case of large lesions involving the whole gonad, the second ROI was
In the case of large lesions involving the whole gonad, the second ROI was drawn on
drawn on normal parenchyma of the contralateral testicle. For each ROI, the software
normal parenchyma of the contralateral testicle. For each ROI, the software generated
generated time–intensity curves to calculate the following parameters: peak intensity (PI,
time–intensity curves to calculate the following parameters: peak intensity (PI, dB), i.e., the
dB), i.e., the maximum intensity of the signal produced by the contrast; time-to-peak
maximum intensity of the signal produced by the contrast; time-to-peak (TTP, s.), i.e., the
(TTP, s.), i.e., the interval from injection (time 0) until PI, and the area under the curve
interval from injection (time 0) until PI, and the area under the curve (AUC). Moreover,
(AUC). Moreover, the washout (WO, s.) was arbitrarily defined as the time interval from
the washout (WO, s.) was arbitrarily defined as the time interval from TTP until signal
TTP untildeclined
intensity signal intensity declined
by 40% of by 40%
PI (Figure 5). of PI (Figure 5).

Figure 5. Representative image showing quantitative CEUS examination with ROIs positioned
Figure theRepresentative
within 5. lesion (yellow image showing
ring) and in the quantitative CEUS examination
normal parenchima with ROIs positioned
(blue ring) respectively on the left
and corresponding
within time-intensity
the lesion (yellow ring) and curves in the right.
in the normal parenchima (blue ring) respectively on the left and
corresponding time-intensity curves in the right.
Animals 2022, 12, 210 7 of 17

2.6. Histopathological Examination

Testicles were submitted for histopathologic examination in 10% buffered formalin
and were then trimmed, embedded in paraffin wax, and sectioned at 4 µm. The sections
were stained with hematoxylin and eosin and examined for evidence of abnormal testicular
parenchyma. Definitive diagnosis of the testicular lesions was made by histopathologic
evaluation reviewed by a single veterinary pathologist blinded to the imaging ultrasound
findings and clinical diagnosis.

2.7. Statistical Analysis

Two off-site radiologists (co-authors P.B. and A.T.) retrospectively reviewed the con-
ventional ultrasonography techniques, Doppler, B-flow, and CEUS images of each dog used
for the statistical analysis. Neither radiologist had performed the US examinations and
both were blinded to the final diagnoses and to any other clinical information. Consensus
was achieved for all variables examined and the final interpretation used for the statistical
evaluation was based on the previously defined diagnostic criteria.
Discrete variables are presented as median and range, while categorical data as
frequency or percent frequency. All quantitative data, evaluated for normality by the
Kolmogorov–Smirnov test, are reported as mean ± standard deviation. The independent
t-test was applied for comparing Doppler and CEUS quantitative parameters between
testicular lesions of each different tumor type and normal parenchyma. The comparisons
between ultrasonographic blood flow parameters of different testicular tumors were tested
using ANOVA followed by post hoc Tukey test for contrast between means. For compar-
isons of categorical data, the chi-sqare test or Fisher exact test were applied. A p value <0.05
was considered indicative of a statistically significant difference.
Agreement between color Doppler, Power Doppler, and B-flow techniques for the
evaluation of testicular tumor blood flow patterns was calculated using Fleiss kappa; 95%
confidence intervals (CIs) are also provided. Kappa values were interpreted as follows:
between 0.21 and 0.40 were considered as slight agreement, between 0.41 and 0.60 as
moderate agreement, while values between 0.61 and 0.80 as substantial agreement. Their
diagnostic sensitivity (true positives/(true positive + false negative)), specificity (true
negatives/(true negative + false positive)), and positive and negative likelihood ratios
(calculated as sensitivity/(1-specificity) and (1-sensitivity)/specificity, respectively) in dif-
ferentiating the tumor types as well as their accuracy were assessed by comparing the
findings with histological results. Based on our own clinical evidence, in the case of leydigo-
mas, true positive cases were lesions characterized by the presence of peri/intralesional
arteries and histological confirmation. True negative cases were lesions characterized by
the presence of intralesional arteries and histological confirmation of tumors other than
leydigoma. False positive lesions were those with the presence of peri/intralesional arteries
and tumors other than leydigoma. False negative cases were defined as lesions with intrale-
sional arteries and histological confirmation of a leydigoma. In the case of seminomas, the
distinctive trait for a positive test was the presence of an intralesional artery and histological
confirmation.
Demographic and specific characteristics of testicular tumors were reported descrip-
tively for all the lesions in the study population. For comparative statistical analysis,
whenever bilateral lesions of the same etiology were found, we included only the data of
the right testicle. Whenever multiple lesions were present in the same testicle, we only
used only values obtained from the greatest lesion among those of the same etiology. All
statistical analyses were performed using commercially available software.

3. Results
3.1. Study Dogs and Reference Diagnosis
Twenty-seven dogs met the inclusion criteria. The dogs ranged in age from 5.5 to
16 years old (median, 11.0 years) and in weight from 5 to 35 kg (median, 26 kg). There
were sixteen mixed breed (MB) dogs, two Doberman pinschers (DP), and one for each of
Animals 2022, 12, 210 8 of 17

the following breeds: American Stafforshire (AS), Basset Hound (BH), Dachshund (DH),
Epagneul Breton (EB), French Bulldog (FB), German shepherd (GS), Italian Mastiff (IM),
Labrador Retriever (LR) and Pointer (PR).
Twelve dogs had unilateral lesions (five on the right and seven on the left testis), while
15 dogs had bilateral lesions. In total, the 42 (20 right and 22 left) pathological testes had
46 tumor lesions, 38 of which were single, including one in a cryptorchid testis and four
were multiple. Testicular lesions (Table 1) included 12 seminomas (26.1%), seven Sertoli cell
tumors (15.2%), 21 Leydig cell tumors (45.7%), and six mixed tumors (13.0%). The mixed
tumors were Leydig cell tumors combined either with Sertoli cell tumors (three lesions) or
with seminoma (three lesions). By B-mode, we identified 45 of 46 lesions.

Table 1. Characterization of the 46 testicular tumor lesions detected in 27 dogs of different breeds
included in the study.

Number of Lesions
Testicular Distribution Site Tumor Type Breeds *
Single Multiple Total
Leydigoma FB 1
Right Sertolioma 2 MB + EB 3 5
Seminoma GS 1
Monolateral
Leydigoma 4 MB + DP 5
Left Sertolioma BH 1 7
Mixed MB 1
Leydigoma 3 MB + IM + DP + AS + DH 7 2
Sertolioma PR 1
Right 17
Seminoma 5 MB + LR 6
Mixed MB 1
Bilateral
Leydigoma IM + DH + MB + DP + AS 5 1
Sertolioma MB + PR 2
Left 17
Seminoma 4 MB + LR 5
Mixed 3 MB 3 1
Total 42 4 46
* See text for codes of dog breeds.

The seminomas (n = 12) were found in eight dogs. The tumors were unilateral in
one dog involving the right testicle and bilateral in four dogs. In the other three dogs,
the lesions (one left and two right) were associated with a different type of tumor in the
contralateral testicle. The Leydig cell tumors (n = 21) were detected in 14 dogs. In six
dogs, the lesions were unilateral involving the left (n = 5) and right (n = 1) testis. In four
dogs, the leydigomas (one left and three right) were associated with a different type of
tumor in the contralateral testicle. In the other four dogs, the leydigomas were bilateral
with double lesions in three testicles. The sertoliomas (n = 7) were found in six dogs. One
sertolioma was not detected using conventional B-mode, Doppler ultrasound, or B-flow
imaging and was revealed only at CEUS. Four dogs had unilateral lesions, one on the left
retained testicle and three on the right testis, one dog evidenced bilateral lesions, while in
one dog the sertolioma were associated with a different type of tumor in the contralateral
testicle. Mixed tumors (n = 6) were found in the testicles of five dogs. Only one lesion
was unilateral on the left testicle, whilst the others appeared in bilateral lesions (with other
non-mixed tumors found in contralateral testes).
No signs of metastasis were identified by ultrasound examination of abdominal lymph
nodes or thoracic X-ray images, with the exception of two dogs. One dog showed the
involvement of aortic lymph nodes (the cytological examination confirmed a metastasis of
a bilateral sertoliomas) while the other had lung metastasis visible through X- ray due to
bilateral seminomas.
Animals 2022, 12, 210 9 of 17

3.2. Conventional Ultrasonography of Testicular Lesions

Testicular lesions included in the comparative analysis (n = 33) had a mean size
of 16.0 ± 10.9 mm, 26 of which (78.8 %) were small enough to leave normal testicular
parenchyma around the lesions (focal tumors), while seven (21.2%) occupied the whole
testes (diffuse tumors). Eleven lesions (42.3%) were rounded and fifteen (57.7%) were
oval-shaped. Out of 33 lesions, 10 (30.3%) were hyper-echogenic, one (3.0%) isoechoic,
and the remaining 22 (66.7%) hypoechoic. In eight cases, cystic structures were evidenced
within the lesions by conventional ultrasonography and confirmed histopathologically.
Seminomas (n = 8) appeared mainly hypoechoic (six in eight, 75.0%) and less frequently
hyperechoic (two in eight, 25.0%), with either round (37.5%) or oval lesions (62.5%), mainly
with irregular margins (seven in eight, 87.5%) and inhomogeneous pattern (five in eight,
62.5%). They ranged in size from 6.6 to 55 mm (19.1 ± 16.2 mm).
Leydigomas (n = 14) were all almost focal lesions (13 in 14, 92.9%), either round (eight
in 14, 57.1%) or oval (six in 14, 42.9%), 4 to 30 mm in size (13.5 ± 8.4 mm). The lesions were
mainly hypoechoic (10 in 14, 71.4%) and hyperechoic (four in 14, 28.6%) to the surrounding
parenchyma with heterogeneous pattern in nine out of 14 (64.3%) and irregular margins in
eight out of 14 cases (57.1%). In three lesions, anechoic cystic-like round structures were
detected.
The sertoliomas (n = 6), ranging from 6 to 25 mm in diameter (16.2 ± 7.0 mm), were
mainly focal lesions (five in six, 83.3%) showing an oval shape (four in six, 66.7%) with
either well defined (three in six, 50.0%) or irregular (three in six, 50.0%) margins. They
showed homogeneous (two in six, 33.3%) or inhomogeneous (four in six, 66.7%) patterns
and hypo/isoechoic/hyperechoic structures in three, one, and two cases, respectively. In
half of the lesions, conventional ultrasonography evidenced anechoic cystic-like round
structures.
The mixed tumors (n = 5) were either focal (60.0%) or diffuse (40.0%) lesions, 5–30 mm
in diameter (17.6 ± 11.4 mm). They were mainly oval shaped (four in five, 80.0%) with
irregular margins (five in five, 100%) and an inhomogeneous pattern (four in five, 80.0%)
with either hypo (three in five, 60.0%) or hyperechoic structures (two in five, 40.0%). In two
lesions, there were anechoic cystic structures. In these mixed lesions, Leydig cell tumors
were associated with seminoma (three cases) or with sertolioma (two cases).
The mean sizes of testicular lesions did not vary between the different tumor types
(F = 0.4743, p = 0.703). Similarly, the type, shape, and margin of lesions as well as their
echogenicity did not differ between tumor types.

3.3. Doppler Ultrasonographic Study

With color Doppler ultrasonography, arteries of the pampiniform plexus and marginal
arteries of testis were easily visualized in all dogs. Using color Doppler ultrasound,
the blood flow pattern was perilesional in eight tumors, intralesional in 15, and both
perilesional and intralesional in five lesions, while there was no blood flow pattern ob-
servable in the remaining five lesions, including three leydigomas, one seminoma, and
one sertolioma (Table 2). Counted jointly, perilesional and perilesional/intralesional pat-
terns, the frequencies of blood flow patterns among testicular tumors varied significantly
(chi-sqare (3, 28) = 9.938, p = 0.0191). The proportion of leydigomas with either a perile-
sional and/or perilesional/intralesional blood flow pattern was higher that of all the other
tumors counted together (two in 11 vs. 13 in 17, p = 0.0056). This resulted in a sensitivity of
81.8%, a specificity of 76.5%, a correct classification rate of 78.6%, and a positive likelihood
ratio of 3.5.
Animals 2022, 12, 210 10 of 17

Table 2. Color Doppler characterization of arterial distribution in different tumor lesions (n = 33)
included in the comparative analysis. The arteries (number and relative frequencies) were categorized
as intralesional, perilesional, intra/perilesional, or absent.

LEYDIG SERTOL SEMIN MIXED

Arteries
n. % n. % n. % n. %
Intralesional 2 14 4 67 6 75 3 60
Perilesional 5 36 0 0 1 13 2 40
Intra/Perilesional 4 29 1 17 0 0 0 0
Absent 3 21 1 17 1 13 0 0
Total 14 6 8 5

Among the pulsed-wave Doppler velocimetry values of pampiniform plexus (n = 33),

PI and RI differed (p < 0.01) between the testicular tumors (Table 3). The PI of sertoliomas
was higher than that of seminoma and mixed tumors (1.59 vs. 0.76 and 0.81, p = 0.0068 and
p = 0.0123, respectively). The RI of seminomas was lower than that of sertoliomas (0.43
vs. 0.66, p = 0.0124). In the marginal artery of affected testicles (n = 33), PI and RI varied
(p < 0.01) between the different tumor types. The mean values of both indexes were higher
in sertoliomas (1.67 ± 0.59 and 0.67 ± 0.11, respectively) than in leydigomas (0.57 ± 0.22
and 0.40 ± 0.14, p = 0.00004 and p = 0.0016, respectively), seminomas (0.50 ± 0.23 and
0.40 ± 0.13, p = 0.00001 and p = 0.00009, respectively), and mixed tumors (0.84 ± 0.63 and
0.49 ± 0.19, p = 0.0006 and p = 0.0245). With this technique, lesion blood flow was sampled
only in about half of the testicular tumors considered for the comparative analysis (16 in
33, 48.5%). In the intra- and/or perilesional arteries, PSV and EDV (Table 3) were higher
in leydigomas (n = 7; 29.5 ± 10.0 and 19.1 ± 6.9, respectively) than in all the other tumors
considered together (n = 9; 16.6 ± 3.6 and 8.6 ± 23, respectively).

Table 3. Pulsed-wave Doppler parameters derived from three different regions drawn within pampini-
form plexus, marginal, and intra-testicular arteries of pathological testicles with different tumor
lesions (n = 33) included in the comparative analysis. Peak systolic velocity = PSV, End diastolic
velocity = EDV, Resistance index = RI, Pulsatility index = PI. Values are means ± S.D.

Doppler LEYDIGOMA SERTOLIOMA SEMINOMA MIXED

Arteries
Parameters Mean ±SD n Mean ±SD n Mean ±SD n Mean ±SD n
PSV 23.4 8 14 23 9.6 6 24.8 14.9 8 18.4 10.9 5
EDV 9.9 4.2 14 6.4 2.8 6 11.8 3.8 8 9 7.5 5
Plexus
PI 1 0.3 14 1.6 0.8 a 6 0.8 0.5 b 8 0.8 0.2 b 5
RI 0.6 0.1 14 0.7 0.1 a 6 0.4 0.2 b 8 0.5 0.1 5
PSV 23.4 13.7 14 23.3 0.6 5 18.1 5.2 8 18.5 10.5 4
EDV 14.5 8.6 14 6.4 3.7 5 11.3 4.3 8 8.5 4 4
Testicular
PI 0.6 0.2 b 14 1.7 0.6 a 5 0.5 0.2 b 8 0.8 0.6 b 4
RI 0.4 0.1 b 14 0.7 0.1 a 5 0.4 0.1 b 8 0.5 0.2 b 4
PSV 29.5 10 a 7 18.7 1.4 b 4 13.1 3b 3 17.9 4.7 b 2
Intra/Peri EDV 19.1 6.9 a 7 10.3 2.4 b 4 7.2 0.6 b 3 7.5 2.3 b 2
lesional PI 0.6 0.1 7 0.7 0.1 4 0.6 0.3 3 0.9 0.63 2
RI 0.4 0.1 7 0.5 0.1 4 0.4 0.1 3 0.6 0.2 2
Different letters within the same row indicate significant differences for p ≤ 0.05.

In five testicular tumor lesions, power Doppler failed to detect any kind of vascular-
ization, while it was perilesional in six tumors, intralesional in 11, and both perilesional
and intralesional in the remaining 11 lesions (Table 4). Counted jointly, perilesional and
perilesional/intralesional patterns, the frequencies of blood flow patterns varied signifi-
cantly among testicular tumors (chi-sqare (3, 28) = 8.714, p = 0.0333). In leydigomas, the
frequency of perilesional and/or perilesional/intralesional blood flow pattern (10 in 12)
Animals 2022, 12, 210 11 of 17

was higher (chi-sqare (1, 28) = 4.504, p = 0.0338) than that of all the other tumor types
combined (7 in 16). This resulted in a sensitivity of 83.3%, a specificity of 56.3%, a correct
classification rate of 67.9%, and a positive likelihood ratio of 1.9. Power Doppler signals of
intralesional and perilesional vessels (Table 4) did not differ between the different testicular
tumors, although their mean values were higher in leydigomas (n = 12; 10,392 ± 9655) than
in seminoma (n = 6; 6192 ± 5486), sertoliomas (n = 5; 5519 ± 3127), and mixed cell tumors
(n = 5; 5519 ± 5115).

Table 4. Power Doppler characterization of arterial distribution in different tumor lesions (n = 33)
included in the comparative analysis and color signals in pixels (mean values ± SD). The arteries
(number and relative frequency) were categorized as intralesional, perilesional, intra/perilesional,
or absent.

Arterial Distribution Power Doppler

Tumor Type Internal Peripheral Both Absent Pixel Values
n. % n. % n. % n. % Mean ±SD n
LEYDIG 2 14 3 21 7 50 2 14 10,391 9655 14
SERTOL 4 67 0 0 1 17 1 17 5519 3127 6
SEMIN 4 50 1 13 1 13 2 25 6192 5265 8
MIXED 1 50 2 40 2 40 0 0 5519 5115 5

3.4. B-Flow Examination

Using the B-flow technique, blood flows were easily imaged in testicles of all dogs and
in all lesions, except one sertolioma. In the comparative analysis, the vascularization type
of lesions was scored as one in 19 (57.6%) and as two in 13 (39.4%) out of 33 tumors. In one
lesion (3.0%), vascularization was scored as 0 (Table 5). The frequencies of vascularization
types did not vary among the different tumor types (chi-sqare (3, 33) = 3.438, p = 0.3294).

Table 5. B-Flow characterization (numbers and relative frequencies) of arterial vascularization

and arterial distribution in different tumor lesions (n = 33) included in the comparative analysis.
The arterial vascularization was categorized as 0, 1 or 2 if absent, smaller or larger than 2 mm in
diameter respectively; the arterial distribution was classified as P if the vessels were perilesional or I
if distributed within the tumor lesion and I/P if both intra/perilesional.

Vascularization Codes Distribution Flow Types

Lesion Type n.
0 % 1 % 2 % P % I % I/P %
LEYDIG 14 0 0 7 50 7 100 6 43 2 14 6 43
SERTOL 6 0 0 4 67 2 33 0 0 4 67 2 33
SEMIN 8 1 13 6 75 0 16 1 13 7 88 0 0
MIXED 5 0 0 2 40 3 60 2 40 3 60 0 0

The vascular patterns of testicular lesions varied between the different types of tumors
(chi-sqare (3, 33) = 12.491, p = 0.0058). In leydigomas, the frequency of perilesional and/or
perilesional/intralesional blood flow pattern (12 in 14) was higher (chi-sqare (1, 33) = 11.507,
p = 0.00317) than that of all the other tumor types (five in 19). In seminomas, the frequency
of an intralesional blood flow pattern (seven in eight) was higher (chi-sqare (1, 33) = 6.435,
p = 0.01118) than that of all the other tumor types considered together (Table 5). This
resulted in a sensitivity of 85.7%, a specificity of 73.7%, a correct classification rate of 78.8%,
and a positive likelihood ratio of 3.3.

3.5. Contrast-Enhanced Ultrasonography

No adverse side effects associated with the intravenous injection of the contrast agent
were observed.
Animals 2022, 12, 210 12 of 17

CEUS identified all testicular tumor lesions, including those not revealed by conven-
tional ultrasonography, Doppler ultrasound, or B flow. Qualitatively, in the comparative
analysis, 21 (63.6%) of the 33 lesions showed an inhomogeneous pattern during the wash-in,
while 12 (36.4%) were homogeneous. The blood flow patterns of wash-in did not differ be-
tween tumor types (chi-sqare (3, 33) = 5.942, p = 0.1153). Eight of 33 lesions (24.2%) showed
a hypoenhanced wash-in phase, whilst 18 (54.5%) were hyperenhanced and the remaining
seven (21.2%) isoenhanced. The frequencies of wash-in contrast enhancement did not vary
between leydigomas and the other tumor types (chi-sqare (2, 33 = 3.6192, p = 0.163723).
Quantitative perfusion parameters of time–intensity curves derived from ROIs po-
sitioned on testicular tumor lesions and normal testicular parenchyma were taken. Peak
intensity and AUC mean values of leydigomas (n = 14) were higher (p < 0.05) compared
to those of paired normal parenchyma (−52.1 ± 6.6 vs. −56.6 ± 4.3 and 542.3 ± 317.2 vs.
362.0 ± 210.0, respectively), while TTP was lower (29.7 ± 16.6 vs. 31.9 ± 18.2 s.). The peak
intensity and AUC mean values of seminomas (n = 8) were higher (p < 0.01) compared
to those of paired normal parenchyma (−52.1 ± 4.7 vs. −57.9 ± 3.3 and 406.4 ± 192.4 vs.
278.1 ± 168.0, respectively), while TTP was lower (p < 0.01; 26.9 ± 4.3 vs. 33.5 ± 8.4 s.).
Peak intensity, TTP, and AUC mean values of sertoliomas and mixed cell tumors did not
vary from those of corresponding normal parenchyma.
CEUS parameters (PI, TTP, AUC, and washout) derived from lesion ROIs did not vary
among the different tumor types.

3.6. Agreement between US Techniques

Taking the three different US techniques (color Doppler, power Doppler, and B-flow)
to be the raters, interrater ultrasonographic agreement, as assessed by Fleiss kappa, showed
substantial agreement (0.64) in identifying intra/perilesional blood flow patterns (95% CI:
0.42, 0.86).

4. Discussion
To our knowledge, this is the first study to evaluate a multiparametric ultrasono-
graphic approach for the diagnosis of testicular tumors in dogs. In principle, an accurate
evaluation of the blood flow of testicular tumors should allow a better characterization of
their etiological nature than that obtainable by conventional ultrasonography alone. Indeed,
color Doppler, power Doppler, or B flow can be used to examine the vascular patterns of
testicular tumor lesions sharing a good rate of agreement. B flow appears to be superior in
terms of sensibility. On the other hand, CEUS allows the detection of all testicular tumors,
including those not identified by the other ultrasonographic techniques used. Despite that,
CEUS did not allow for the differentiation of any testicular tumor type.
In the present study, there was a higher prevalence of dogs with Leydig cell tumors
in accordance with previous reports [24,25]. Conversely, in other recent reports [4,26,27],
seminomas had the highest incidence. In our opinion, these differences are likely due to
different criteria for enrolling dogs and the variable number of cases included in each study.
Conventional B-mode ultrasonography is considered a reliable, non-invasive tech-
nique, valuable for the detection of testicular tumors [8], even though their imaging features
are not useful for the characterization of any particular tumor type [9]. In our study, 2D
ultrasonography was able to spot testicular tumors in all dogs, except a Sertolioma in
the testicle of a Basset hound. In agreement with previous studies [9,28], the ultrasound
characteristics of tumors evaluated in grayscale, such as size, shape, and margins, were
extremely variable, making their appearances inconsistent for differentiating any tumor
type. In particular, there was a prevalence of focal lesions compared to the diffuse lesions
occupying the entire testicle. The lesions showed a mainly hypoechoic structure with no
peculiar characteristic that could differentiate them based on tumor type.
Color Doppler ultrasonography, pulsed wave spectral Doppler, and power Doppler
applications are all reliable techniques for evaluating blood flow perfusion in canine
reproductive tracts in different physiological and pathological conditions [11,29–31]. Color
Animals 2022, 12, 210 13 of 17

and pulsed Doppler ultrasound can assess not only the presence and direction of the blood
flow, but also its dynamic parameters. Color and pulsed Doppler ultrasonography with a
high-frequency transducer represent the imaging of choice for the initial identification of
suspected neoplastic testicular lesions in human males [32]. When using pulsed Doppler
ultrasonography, however, careful interpretation is required to evaluate blood flow velocity
because such a parameter depends on the beam angle insonating the vessel. Conversely,
this limiting factor does not influence resistive and pulsatility indices: the first reflects the
resistance to blood flow caused by microvascular beds distal to the site of measurement,
while the other quantifies the pulsatility or oscillations of the waveform during a cardiac
cycle. For these reasons, PI and RI are widely used to assess testicular blood flow perfusion
in humans and animals in physiological [12,13,33–36] and pathological conditions [11,37,38].
Compared to color and pulsed Doppler, power-Doppler imaging has a major sensitivity
even for slow blood flow. In addition, power Doppler mode can improve the estimation
of blood flow even in vessels with small diameters and weak blood flow because it is not
influenced by the insonating angle [39]. However, despite the proven usefulness of Doppler
ultrasonography, to our knowledge, there are only two papers regarding the use of these
imaging techniques for the diagnosis of testicular tumors in dogs [11,31].
Color Doppler ultrasonography failed to identify perilesional and/or intralesional
vascularization in five out of 33 testicular tumors included in the comparative analysis,
but one should keep in mind that this technique has limited resolution and may fail to
identify blood flow in small vessels and/or lesions with poor blood flow. In the present
study, leydigomas had a perilesional or combined peri/intralesional distribution patterns
in 81.2% of the cases, while the other types of tumors had a mainly intralesional vascular-
ization pattern. However, the distinction between intra and/or perilesional vessels cannot
differentiate among the other types of testicular tumors, such as seminomas, sertoliomas, or
mixed cell tumors. Thus, the recognition of distinctive vascular features with color Doppler
ultrasonography has quite limited usefulness for the differential diagnosis of testicular
tumors in dogs being greatly influenced by their prevalence. Bigliardi et al. [31] reported
similar findings using color Doppler imaging in dogs with different neoplastic lesions.
In our study, pulsed Doppler allowed the examination of pampiniform plexus in the
testicles of all dogs. While peak systolic and end diastolic velocities of the pampiniform
plexus did not vary between testicles with different tumors, the resistance index as well
as the pulsatility index were significantly higher in testicles with sertoliomas than in
testicles with seminoma. By pulsed Doppler, the marginal artery was sampled in all lesions
included in the comparative analysis. Once again, peak systolic and end diastolic velocities
of the marginal arteries did not vary between testicles with different tumors, but both
resistance and pulsatility indexes in sertoliomas were higher than in the other types of
tumors. Taken together, these findings are difficult to interpret given that both resistance
and pulsatility indexes are ratios of two other primary variables (i.e., peak systolic velocity
and end diastolic velocity) which did not vary between the different tumor types. Pulsed
Doppler identified some perilesional and/or intralesional vascularization only in 16 out of
33 testicular tumors. Nevertheless, leydigomas were characterized by higher mean values
of PSV and EDV than those of the other tumors considered together. These results suggest
that Leydig cell tumors may influence the vascular architecture of the lesion and/or its
functional characters. However, these findings should be taken with caution due to the
small number of tumors with detectable vascularization, making comparisons between
each tumor type poorly reliable. Based on the present data, therefore, the potential ability
of pulsed Doppler in differentiating the nature of testicular tumors remains questionable
mainly due to the poor resolution of this technique. Similar results were reported by
Gunzel-Apel et al. [11] and Bigliardi et al. [31] in dogs. Gunzel-Apel et al. showed that all
tumors examined (n = 5) had a different blood flow pattern compared with normal testes,
but they did not demonstrate any difference between testicular tumors. Somewhat similar
results were reported by Bigliardi et al. [31], since they found an increasing perfusion
of neoplastic testicular parenchyma (that accounted for 40 in 50) of all testicular lesions
Animals 2022, 12, 210 14 of 17

examined, with significantly higher values in tumor lesions. Moreover, they described
a 40–50% increase of the resistive index of neoplastic lesions compared to RI values of
non-neoplastic lesions.
In our study, power Doppler allowed the visualization of blood flow in 28 out of 33
tumor lesions without any improvement in the detection limit compared to color Doppler.
Regarding the vascular patterns of testicular tumors, power Doppler confirmed the ob-
servation recorded with color Doppler with a predominant perilesional or combined
peri/intralesional distribution pattern in leydigomas and an intralesional vascularization
pattern in the other types of tumors. Similarly, based on the characterization between intra
and/or perilesional vessels, power Doppler cannot differentiate among the other types of
testicular tumors such as seminomas, sertoliomas, or mixed cell tumors. Quantitatively, the
number of color pixels detected by power Doppler did not vary between testicular tumors.
B-Flow is based on the GE-patented, digitally encoded ultrasound technique that was
introduced in high-frequency transducers several years ago to digitally suppress unwanted
signals (e.g., noise and tissue) and boost weak signals (e.g., blood echoes) to overcome
some limitations of Doppler [40]. This technique visualizes blood flow echoes in gray scale
imaging with different gray intensities according to the reflector’s speed and dynamics.
This technology allows the direct visualization of intravascular flow echoes during real-
time gray-scale ultrasonography. In addition, the technique is relatively simple to operate,
with fewer parameters to manipulate than color Doppler. B-Flow imaging is very sensitive
and in one study of placenta blood flow in women it allowed the visualization of a larger
number of small (less than 5 mm) vessels compared to color Doppler [41]. In our study, B
flow allowed the detection of the vascularization patterns in all the lesions considered for
the comparative analysis. The vascular distribution pattern considering the intralesional
vascularization alone against the perilesional alone or peri-intralesional combined varied
significantly among the different tumor types. As for color and power Doppler, with B flow
there was a predominant perilesional or combined peri/intralesional distribution pattern
in leydigomas and an intralesional vascularization pattern in the other types of tumors.
Similarly, based on the different patterns of intra and/or perilesional vascularization, B
flow cannot differentiate among the other types of testicular tumors such as seminomas,
sertoliomas, or mixed cell tumors. On the contrary, the distribution of frequencies of arterial
sizes did not vary among the different tumor types. As far we know, this is the first study
to examine the vascularization of testicular tumors in dogs with B flow. Quite surprisingly,
in human medicine, the B Flow sonographic technique is not used very often in testicular
tumor diagnosis, despite being much more sensitive than color Doppler.
No dog experienced adverse reactions to the injection of contrast agent. Once again,
our clinical findings add further support to the safety of CEUS as documented by many
previous studies [42–45]. As said, CEUS identified all tumors, including the sertolioma
that was not detected by the other ultrasonographic techniques including B flow imaging.
Independent of the testicular tumor type, in fact, at least one or more of the parame-
ters characterizing the distribution and/or the pattern of the contrast within the lesion
greatly differed from that of normal surrounding testicular parenchyma. In leydigomas
and seminomas, mean quantitative parameters (PI, TtP, and AUC) derived from time–
intensity curves of the lesion ROIs significantly differed from those obtained from normal
parenchyma. Conversely, sertoliomas and mixed cell tumors did not show any such dif-
ferences. However, the same quantitative parameters as well as the qualitative ones did
not vary between the different tumor types. Therefore, based on our results, CEUS cannot
be considered the diagnostic test of choice to differentiate the etiology of testicular tumors
in dogs. These results are in agreement with those reported in human medicine litera-
ture [46,47] but partially disagree with those found in dogs by Volta et al. [7]. The latter
authors, in fact, reported that hypo or isoenhanced testicular lesions with intralesional
vessels were associated to seminomas, thus allowing a partial differentiation between
testicular tumors.
Animals 2022, 12, 210 15 of 17

All ultrasonographic techniques employed here (color and power Doppler, B flow,
and CEUS) examined in real time the vascular perfusion in the same testicular tumors, each
one with its own potential diagnostic potential due to technical features and underlying
software. Interestingly, while CEUS did not contribute to the differential diagnosis of
testicular tumors, it was highly effective in their detection by revealing an extra lesion.
However, we cannot exclude that CEUS could have an even greater sensitivity in spotting
small tumors. In fact, the marginal contribution of CEUS to the identification of testicular
tumors may have been influenced by the enrolment criterion based only on the presence
of a lesion identified in B-mode. Pulsed Doppler evidenced some differences in blood
flow velocimetry of the pampiniform plexus, testicular arteries, and intralesional arteries
between the different tumor types. However, in the latter case, diagnostic performance was
greatly hampered by its poor sensibility in imaging blood flows, which made this technique
inapplicable in almost 50% of testicular tumor lesions. There was some relationship between
the presence or absence of intralesional and/or perilesional vascularity as assessed by
Doppler or B-flow signals and the different types of tumors. Certainly, compared to color
Doppler and power Doppler, B-flow was more effective in detecting blood vessels in and/or
around testicular lesions, even if these ultrasonographic techniques showed a good rate of
inter-agreement.

5. Conclusions
In conclusion, the present work indicates that testicular tumors of dogs have subtly
different vascular patterns, a condition that could help in identifying their etiology. To
this end, a multiparametric approach with the simultaneous use of color Doppler, B-flow,
and CEUS ultrasonography is highly recommended. Despite the fact that our results are
somewhat preliminary due to the limited number of dogs enrolled, we believe that these
techniques could be useful to improve the detection of testicular tumors and to investigate
clinical problems of hypo/infertility of male dogs. This would be particularly useful in
those cases where a unilateral castration could be suggested, thus preserving future fertility,
or where the presence of a tumor with low metastatic incidence may suggest skipping
surgery and avoiding any anesthesiological risk.

Author Contributions: R.O. and E.V. equally contributed to the conceptualization of this study,
drafted the clinical protocol, and performed the clinical investigations. C.B. supervised and analyzed
the data. P.B. and A.T. critically interpreted the clinical data and drafted the paper. A.P. reviewed and
edited the manuscript. All authors have read and agreed to the published version of the manuscript.
Funding: This research received no external funding.
Institutional Review Board Statement: Not applicable because the dogs were observed following
normal diagnostic procedures for clinical assessment of suspected pathological conditions of testicles.
Informed Consent Statement: Informed consent was obtained from all subjects involved in the
study.
Data Availability Statement: The data presented in this study are available on giustified request
from the corresponding author.
Conflicts of Interest: The authors declare no conflict of interest.

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